Report on the first detection of Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) in the Republic of Benin, West Africa

The Asian citrus psyllid (ACP), Diaphorina citri, was detected for the first time in the Republic of Benin, West Africa. The ACP is a known vector of Candidatus Liberibacter asiaticus (CLas), the putative causal agent of the devastating Huanglongbing (HLB; citrus greening disease). During visual surveys, ACP was only observed on residential citrus trees in southern Benin, but not in residential areas or commercial groves in the central and northern parts of the country. Its identity was confirmed morphologically and molecularly via DNA barcoding with published primers. Analysis of the obtained sequences showed that the ACP recorded in Benin clustered with the ones previously reported from Nigeria, suggesting a common origin of both populations. The ACP samples from Benin also carried Ca. Carsonella ruddii and Ca. Profftella armatura, two commonly found ACP endosymbionts. However, all the sampled ACP individuals tested negative for Ca. Liberibacter africanus, Ca. Liberibacter americanus, and CLas by quantitative polymerase chain reaction. This is the second report of the ACP in West Africa after Nigeria, the eastern bordering country of the Republic of Benin. Benin has an expanding commercial citrus industry, especially in the southern part of the country. Although the ACP samples tested negative for the HLB associated bacteria, the detection of ACP in the country requires swift actions including area-wide surveys to determine the extent of spread of this pest and the implementation of eradication or control efforts to prevent its establishment and spread of HLB in the country.

In the Republic of Benin, citrus is grown commercially as a cash crop and as backyard tree like in many other parts of the world. Commercial citrus production in the country started soon after the independence in the 1960s, with the creation of the national fruits and vegetables company (Société Nationale des Fruits and Légumes, SoN-aFeL) via a Benin-Israeli cooperation 1 . From 1960 to the 1970s, several citrus varieties of lime, lemon, grapefruit, mandarin, sweet oranges and tangelo were introduced for the establishment of commercial groves mainly in the southern part of the country 2-5 , but sweet orange remained the dominant commercial citrus species in the Republic of Benin to date 6 .
From the onset of commercial citrus industry in Benin, the planted acreage slowly increased and reached ca. 2500 ha of sweet oranges in 1977 where it plateaued until 1989. Commercial citrus acreage declined from 1989 to 1992, as a consequence of the closure in 1986 of SoNaFeL because the country underwent the World Bank and International Monetary Fund structural adjustment programs 7 . Subsequently, the harvested commercial orange acreage has rebounded and gradually increased reaching ca. 6500 ha in 2020 6 . Despite the apparent expansion in acreage in recent years mainly due to the implantation of a processing plant in the commercial citrus belt of Benin, citrus yields in Benin remain among the lowest in the world. In 2020, average orange yields were estimated at 2.5 MT ha −1 , the second lowest commercial yields recorded in Africa and far below the world average of 14-15 MT ha −16 . Several biotic and abiotic factors are responsible for these meager citrus yields in Benin. Firstly, commercial citrus groves in Benin have no irrigation systems and trees are only rain fed. Erratic rainfall can

Materials and methods
Sample collection. A citrus pest survey was conducted at six residential sites and three commercial groves in December 2021 in five of the 12 departments of the Republic of Benin to assess the presence of D. citri and T. erytreae ( Table 1). The residential sites were visited upon homeowners' approval to evaluate the health of trees because of either poor growth or the presence of sooty mold. The commercial groves located in close proximity of the main highway were surveyed and sampled during a road trip. At each site, the host plant species was determined by evaluated the leaves and/or fruit when present, and the tree or grove location coordinates were recorded using the Compass app of an IPhone 12 pro 32 . All residential host plants present at the survey sites were visually examined for the presence of arthropod pests with special reference to the life stages of ACP or their wax-like feeding droppings on leaves 33 and of ACT and the small pit galls it causes on young leaves 34 . In commercial groves, a sample of ten trees was randomly selected for evaluation. As young flush shoots are the breeding sites for ACP and ACT 34,35 , these flush shoots when present, were preferentially examined. Suspect ACP nymphs and adults were collected and stored in plastic vials containing 95% ethanol until further analysis and processing. No ACT was collected during the surveys, hence subsequent analyses focused on ACP.
Identification of D. citri. Morphological identification. All insect samples were brought to the Texas A&M University-Kingsville Citrus Center (TAMUK-CC) entomology laboratory for further analysis. Using the morphological characteristics described by Mead 33  Nucleic acid isolation and PCR. Total nucleic acids were extracted from morphologically identified D. citri adults and nymphs following the Dellaporta et al. 37 protocol. A NanoDrop 2000 series spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to quantify and analyze the quality of the extracted nucleic acids. After quantification and quality analysis, the total nucleic acid samples were stored at − 20 °C until when they were subjected to polymerase chain reaction (PCR) assays as described by Oke et al. 28 . Briefly, a 2 µL aliquot of each total nucleic acid sample was used as template in a 25 μL PCR with reagents and Rapid Protocol described for the PrimeSTAR GXLDNA Polymerase (Takara Bio USA, Inc., Mountain View, CA). PCR was performed on each insect total nucleic acid sample using three distinct primer pairs that target specific genes encoded by the ACP and two of its endosymbionts. The 834 bp fragment of the mtCOI coding region was targeted by the DCITRI COI-L (5′-AGG AGG TGG AGA CCC AAT CT-3′) and DCITRI COI-R (5′-TCA ATT GGG GGA GAG TTT TG-3′) primer pair 38  Bioinformatic analysis. The raw sequences were trimmed to remove the pJET1.2 flanking multiple cloning site sequences with the VecScreen (https:// www. ncbi. nlm. nih. gov/ tools/ vecsc reen/). A consensus sequence from each of the sample-specific forward and reverse sequences was derived using the CAP contig assembly program of the BioEdit software 39 . The derived sequences were scanned with the National Center for Biotechnology Information (NCBI) GenBank database using the BLASTN program 40 for species identification purposes. The MUSCLE alignment program 41 was used to perform multiple sequence alignments for gene-specific datasets of sequences derived from samples analyzed in this study and corresponding sequences of taxon representative retrieved from GenBank. The gene-specific alignment files were used to determine the sequence identity matrices and for phylogenetic analysis with the maximum likelihood algorithm of the molecular evolutionary genetics analysis (MEGA) software version 7.0 42 . in Benin were assayed for the presence of CLaf, CLam and CLas using the Taqman multiplex real-time PCR assays as reported by Oke et al. 28 . Known positive and negative control DNA samples, and non-template water control were included in the reactions. All samples with a cycle threshold (Ct) ≤ 37 were considered positive for a specific target bacterium.

Results
ACP detection and morphological identification. Nine locations (residential sites = 6 and commercial groves = 3) were surveyed during the study (Table 1). Five major ACP host plants including grapefruit, lime, orange jasmine, sweet orange and tangerine were identified at the various locations. Suspect ACP samples were only collected at three residential sites in southern Benin, and none was found in commercial groves and in residential sites in the central and northern departments. The comparison of morphological features of collected samples to voucher specimens 33,36,43 led to their identification as D. citri (Fig. 1). ACP was recovered from three host plants including lime, orange jasmine and sweet orange during the study. While adults were collected from three sites, nymphs were only found on sweet orange trees at one location (Table 1). Considering the spatial separation (2-10 km) of the three detection sites in southern Benin, D. citri may likely be established in this part of the country. During the surveys, aphids and soft scales, including the brown soft scale and the cottony cushion scale, were the most dominant pest species found infesting trees, and they are very likely responsible for sooty mold occurring on the trees.

Molecular detection.
A representative subset of 10 randomly collected ACP (4 adults and 6 nymphs) from the three positive detection sites was selected for molecular analysis. Gene-specific DNA amplicons of the expected sizes were obtained from these samples. Ten mtCOI-specific (GenBank accession no. OP414612-OP414621), 8 argH-specific (OP414446-OP414453) and 10 atpA-specific (OP414454-OP414463) sequences were derived. Analysis of these sequences using BLASTN produced highly significant matches (≥ 99% nt identity; 100% query coverage; E-value 0.0) to corresponding gene-specific sequences of D. citri, Ca. Carsonella ruddii and Ca. Profftella armatura, respectively available in GenBank. In pairwise comparisons, the mtCOI sequences derived in this study shared 99-100% nt identities among themselves and the same range of nt identities with corresponding global sequences of D. citri, indicating that they belong to this psyllid species. As expected, the derived mtCOI sequences from the Benin insect samples were significantly distinct from (and shared only 42-43% identity with) the corresponding sequences of T. erytreae isolates retrieved from GenBank.  www.nature.com/scientificreports/ Phylogenetic analysis. The reconstructed mtCOI maximum likelihood (ML) phylogenetic trees showed the clustering of sequences from Benin within the D. citri clade and distinct from the T. erytreae clade ( Fig. 2A). When the ML tree was reconstructed with only D. citri mtCOI sequences, they clearly segregated into the previously defined Western and Eastern clades 20 with strong (> 50%) bootstrap support (Fig. 2B). All the adult and nymph mtCOI sequences from Benin (OP414612 to OP414621) clustered into the Western clade (Fig. 2B). The argH-specific sequences of the P-endosymbiont Ca. Carsonella ruddii also segregated into the previously defined Western and Eastern clades 46 with strong (> 60%) bootstrap support (Fig. 2C, Supplementary table). Like the ACP mtCOI sequences, all the argH sequences from Benin (n = 8; GenBank acc. Nos. OP414446-OP414453) also clustered into the Western clade (Fig. 2C). In contrast to the mtCOI and argH sequences, the atpA sequences of the S-endosymbiont Ca. Profftella armatura segregated into three distinct clades with strong (> 60%) bootstrap support (Fig. 2D, Supplementary table). Interestingly, all the atpA sequences from Benin (n = 10; GenBank acc. Nos. OP414454-OP414463) segregated into the previously defined ' African' clade, distinct from the Western and Eastern clades (Fig. 2D). This ' African' clade was derived from atpA gene of Ca. Proftella armatura detected in ACP collected in Nigeria 28 . Taken together, the results showed that the field-collected psyllids in Benin and their bacterial endosymbionts are genetically uniform and belonged to the species Diaphorina citri. All the analyzed psyllid samples were negative for CLaf, CLam and CLas by qPCR.

Discussion
Using both morphological identification and molecular tools, our study confirmed the suspect specimens collected in Benin to be D. citri. While D. citri has long been recognized as an invasive pest 22 , this is to the best of our knowledge of current literature, the first report of its detection in Benin. The presence of ACP on the African continent was previously reported from countries in East Africa including Tanzania 21 , Kenya 26 and Ethiopia 27 , and in Nigeria in West Africa 28 (Fig. 3). The current detection in Benin shows that the geographic range of ACP is expanding on the continent, possibly through the increased trade among countries, movement of citrus transplants by hobbyist growers, or natural dispersal. However, its presence in only three residential sites in the south suggests that its spread may be currently limited in the country. Surprisingly, the ACP was detected feeding on three of the most suitable host plants 47,48 in the coastal part of the country with altitude less than 20 m above sea level (Table 1). It is well established that higher altitude limits the incidence of the ACP with its population levels decreasing at higher altitudes probably as a result of differential temperature, air pressure, oxygen level, ultraviolet light or their combinations 49 . However, all sampling sites were below 600 m above sea level, the cut-off point above which no ACP was collected in Puerto Rio 49 . The limited distribution of the ACP in Benin opens avenues for effective management and the enactment of strict quarantine regulations to prevent the incursion of D. citri into the commercial citrus groves mostly located in southern and central parts of the country. The prevailing hot and humid conditions in Benin with temperature ranging from 28 to 32 °C all year round, indicate that the whole country is suitable for the establishment and development of D. citri. Indeed, using a temperature-based model of suitability, Taylor et al. 50 reported that Africa has climate suitable for the establishment of D. citri and HLB. Considering the biology and ecology of D. citri, Aidoo et al. 51 used Maximum Entropy (MaxEnt) to predict that all citrus production areas in Africa are suitable for its establishment although the actual suitability varies with regions. Under current climatic conditions, Benin lies from the high suitability area in the southern and central parts of the country to the medium suitability area in the north 51 , indicating that the whole country is at high risk of D. citri and HLB invasion. The commercial citrus belt is located in the southern part of the country that is deemed highly suitable for D. citri reproduction and development and only situated ~ 100 km away from the current detection sites. This close proximity of the commercial citrus production area to the current D. citri detection sites represents a grave menace to the sustainability of citriculture in Benin. Citrus production in Benin is already crippled by a myriad of biotic and abiotic constraints and a lack of agrochemical inputs. The invasion of D. citri and possible HLB introduction will further reduce yields that are already among the lowest in the world 6 . Hence, a rapid response plan needs to be implemented at the national level, and perhaps regionally across West Africa, to limit the spread of this invasive pest and to control its population within currently established areas.
To gain more insights into the biodiversity of this quarantine significant pest, we analyzed the mitochondrial cytochrome oxidase (mtCOI) gene of specimens collected in Benin. Not only that the mtCOI is versatile because of its high prevalence in cells relative to nuclear genes, it is a highly conserved and maternally inherited gene that is independent of life stages, polymorphism and gender 52,53 . The mtCOI gene has been used with great success in previous D. citri population studies 20,28,38,54,55 . Similarly, the gene-specific sequence of the primary (Ca. Carsonella ruddii) and secondary (Ca. Profftella armatura) endosymbionts of D. citri were used to evaluate their diversity. All the analyzed D. citri individuals from Benin and their primary endosymbiont (Ca. Carsonella ruddii) formed a homogenous group and clustered within the Western clade that consists of psyllid populations that generally include individuals from Asia and the Americas 46,56,57 . However, all the atpA sequences from Benin segregated into the previously defined ' African' clade derived from D. citri collected from neighboring Nigeria, and distinct from the Western and Eastern clades 28 . These results highly suggest that the D. citri samples collected from Benin and Nigeria may be of the same origin and given the close proximity of both countries (Fig. 3), it is highly plausible that this pest may have spread from one country to another. Moreover, these results points to the fact that D. citri may also have been present for quite some time in Benin for local adaptation of the secondary endosymbiont to occur as previously hypothesized 28 . Such local adaptation of Ca. Profftella armatura may be warranted due to its role as a defensive endosymbiont against natural enemies in insects 45 .
The presence of sooty mold on foliage and fruit of the surveyed host plants was the precursor of this study. Sooty mold is generally a cosmetic problem on fruit, but its heavy presence as observed on some leaves can impair photosynthesis, leading to poor tree growth and productivity. This is because the black coating of the sooty mold fungi on leaves could intercept sunlight and perturb leaf temperature and transpiration, subsequently affecting www.nature.com/scientificreports/ the water balance of trees 58 . Although, the ACP was found at one location where sooty mold was recorded on plants, the main culprits of this symptom were soft scales (brown soft scale and cottony cushion scale), aphids and whiteflies that were abundant throughout the country, indicative of poor pest management in citrus production in Benin. In addition, at that site both ACP nymphs and adults were recorded, probably because of the presence of young shoots on sweet orange trees ( Table 1). The ACP is known to reproduce exclusively on young shoots 35 that were absent in most locations at the time of sampling. Increasing citrus production in Benin will require inputs of agrochemicals, irrigation, and effective locallyadapted integrated pest and disease management (IPDM). The detection of D. citri in Benin poses another significant challenge for the sustainability of citrus production, especially if D. citri were to acquire Candidatus Liberibacter asiaticus, causal agent of HLB. Insights gleaned from other invaded citrus producing areas of the world indicate that HLB detection always (and most often surely) lag D. citri detection 18,19,59 . One major foundation for the development of an effective IPDM program will be an understanding of the identity and population dynamics of pests and disease cycles in commercial citrus groves. With the worldwide spread of invasive species, area-wide surveys need to be frequently conducted for early detection of any introduced pest and disease and subsequent implementation of eradication or mitigation approaches.
This work represents a limited and quick study to assess the presence of psyllid vectors of HLB in Benin. The positive detection of D. citri following that of neighboring Nigeria in 2020 28 calls for extensive and regional field surveys to determine the extent of its spread across West Africa and especially in the commercial citrus production areas. Although all the D. citri samples analyzed in this study and those from Oke et al. 28 tested negative for the HLB bacterium, it will be important to continue to systematically survey, sample, and test D. citri from the study sites and surrounding areas since CLas detection in psyllids often predates detection in trees 59 . In conclusion, the current restricted distribution of D. citri in Benin offers an opportunity for limiting its spread and effectively controlling its populations ("Supplemantary information").